Phi Sigma Presentation
- 1. Investigationof the cell-countingmechanism responseto the “large-cell”phenotypein Drosophilamelanogaster Phi Sigma Symposium Presentation Bryston Nham Advisor: Leslie Saucedo, Ph.D Spring 2015
- 2. Normal Development of a Cell (Conlon and Raff, 1999) •Cell Growth •Cell cycle progression •Terminal differentiation •Apoptosis
- 3. Normal Development of a Cell (Conlon and Raff, 1999) •Cell Growth •Cell cycle progression •Terminal differentiation •Apoptosis
- 5. Cell – Counting Mechanism • Recent Studies • Problem Abnormally large Amount of cells Apoptosis initiated to decrease tissue size Large-cell Phenotype: Normal amount of cells Apoptosis not initiated to decrease tissue size
- 6. Big Biological Question • Can abnormal tissue growth from the large-cell phenotype be suppressed by forcing these cells into mitosis? • Could an increase the number of cells possibly alert the cell-counting mechanism? Pathways and Genes of Interest: Rheb-gene, myc-gene, and PI3K pathway.
- 7. Genes and Pathways of Interest • Rheb/Myc-genes and the PI3K pathway • Overexpression = increase in cell mass with failure to promote entry into mitosis. • CDC25 or stg (Drosophila melanogaster) • Phosphatase that activates mitotic cyclin- dependent kinases, which force cells into mitosis.
- 8. +Rheb,myc,PI3K+Rheb,myc,PI3K +stg Cell-Cycle Diagram No entry in mitosis
- 9. = Cell Death (Apoptosis) Normal Tissue +Rheb,Myc, or PI3K +Rheb,Myc, or PI3K +stg Cell-Counting Mechanism
- 10. Protocol Performed • UASGal4 driver system: localizes phenotypic effects to certain region • enGal4: posterior compartment of wing • Four sets of crosses: • Wing tissue analysis based on two characteristics: • 1. Size of the posterior compartment • 2. Cell density in each compartment. • Caspase stain to potentially identify apoptosis. Wild-type x enGal4 x enGal + stg Rheb, myc, or PI3K x enGal4 x enGal + stg
- 12. Results: Control Crosses enGal4 Anterior Posterior enGal4 (+stg) Anterior Posterior
- 13. Results: Rheb-gene crosses enGal4,Rheb Anterior Posterior enGal4,Rheb (+stg) Anterior Posterior
- 14. Results: PI3K crosses enGal4, PI3K Anterior Posterior enGal4,PI3K (+stg) Anterior Posterior
- 15. Results: Myc-crosses enGal4,myc Anterior Posterior enGal4,myc (+stg) Anterior Posterior
- 16. Figure 4. The introduction of stg to cells overexpressing growth pathways increased the cell density within the posterior compartment. Cell density within a surface area of 40 μm2 in the posterior and anterior compartments. When stg is expressed the cell density in the posterior compartment is very similar to the anterior compartment, indicating an increase in mitotic activity. 0 10 20 30 40 50 60 70 80 enG4,WT enG4/stg,WT myc myc (+stg) Rheb Rheb (+stg) PI3K PI3K (+stg) CellCount Posterior Anterior
- 17. Interpretation of Results • Overexpression of gene or pathway caused an increase in cell size and overall tissue size. • Cells did not enter mitosis • When stg was introduced to each cross, tissue size decreased, but cell count increased. • Forced entry in mitosis may have alerted cell- counting mechanism.
- 18. Ongoing and Future/Summer Work • Continue antibody staining • Negative controls showed no apoptosis. • Do antibody staining for stg-crosses. • Verify apoptosis is biological process for decreased tissue size. • Inhibit apoptosis to see growth patterns. • Look at tumor development from a different biological approach • Mutate a few cells rather than entire region.
- 19. References • Chen BJ, Wu YL, Tanaka Y, Zhang W (2014) Small Molecules Targeting c-Myc Oncogene: Promising Anti-Cancer Therapeutics. International Journal of Biological Sciences 10(10); 1084-1096. • Gao Y, et al (2000). Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature 404: 897- 900. • Dang CV. (2012) Myc on the Path to Cancer. Cell Press 149 (1): 22-35. • Cantley, L. (2002) The Phosphoinositide 3-Kinase Pathway. Science 296 (1655). • Shaw, RJ., Cantley, LC. (2006) Ras, PI(3)K and mTOR signaling controls tumour cell growth. Nature 441: 424-430. • Nilsson, I., Hoffmann, I., (2000) Cell cycle regulation by the CDC25 phosphatase family. Prog Cell Cycle Res. 4: 107-114.
- 20. Thank you for your time! Questions?